The ability to rapidly distinguish non-sialated proteins from sialated proteins in a sample is vital to the management of various pathologies; such as; early detection of cerebrospinal fluid (CSF) leakage, endometriosis, melanoma, breast cancer, oral cancer, Alzheimer's disease and similar sialated/non-sialated protein or tau-protein diseases. CSF leakage refers to the escape of the fluid that surrounds the brain and spinal cord, and is caused by rupture of the membrane which surrounds the brain and spinal cord and contains the CSF. Head trauma is responsible for 50-80% of all cases of CSF leakage. Each year in the United States, over one million people are treated and released from hospital emergency departments with traumatic head injuries. Approximately 2 to 3% of head injuries result in the occurrence of CSF leaks; thus this is a relatively common condition seen in hospital emergency rooms. CSF leakage may also result from certain head, spine or brain surgeries, spinal taps, or accidental punctures during neural blockade. The main complication of CSF leakage is cerebral infection (meningitis and brain abscess), which occurs in 25-50% of cases. The prompt initiation of treatment with antibiotics and/or surgical treatment of fistulas is necessary to avoid severe complications. Appropriate treatment therefore requires a rapid and reliable method for determination of CSF leakage.
Radiologic methods, such as CT and MRI scans, can diagnose fractures and detect fluid levels within sinuses, thus suggesting, but not confirming cerebrospinal fluid leakage. Other radiologic techniques include contrast and radionuclide cisternography. Contrast cisternography can locate the site of leakage but may not detect intermittent leaks. Radionuclide cisternography is expensive, has a high false positive rate, and is not accurate in determining the location of leakage. Exploratory surgery, involving the detection of preoperative intrathecally applied fluorescein, is highly sensitive, but this invasive method may also entail some risks to the patient. Measurement of protein and glucose concentrations, such as glucose dipstick tests, have been used in the past as indicators for the presence of CSF. These methods are safe and non-invasive, but have poor reliability.
Beta-2-transferrin is a non-glycosylated (asialated) isoform of transferrin found only in cerebrospinal fluid, ocular fluids, and perilymph. The technology is still unavailable to raise antibodies to target and differentiate between glycosylated and non-glycosylated epitopes. Beta-2-transferrin may be separated from the sialated beta-1-transferrin isoforms and detected by immunofixation electrophoresis using antibodies that bind all transferrin isoforms. With a sensitivity of 94%-100%, and a specificity of 98%-100%, this assay has become the gold standard in detection of CSF leakage. However, because immunofixation electrophoresis is necessary to detect beta-2 transferrin, the assay is expensive ($230-300/sample), and carries multiple added costs for specimen handling, archiving, shipping and storage. Each sample must be provided in high volume, requiring as much as 1-2 ml of sample per assay. Moreover, special technical skills and experienced technicians are required to assure test precision and reliability, mandating that beta-2 transferrin assays be performed by specialty laboratories. As a result, turn-around time for results to reach the caregiver may take up to 4 days, often resulting in additional hospitalization time for the patient suspected of having a cerebrospinal fluid leak, who must remain in the hospital for monitoring until the test results are determined.
Endometriosis is a medical condition that affects 2 to 5 percent of all women. Cells lining the uterus or endometrium grow in other areas of the body, causing pain, irregular bleeding, and even infertility.
Currently, the only accurate diagnosis of the disease requires a laparoscopy, a procedure involving a “belly-button cut” and insertion of a lighted instrument into the navel. However a less invasive method would be the accurate and rapid diagnosis using the methods and the device described herein. Antibodies to the transferrin protein and α2-Heremans Schmidt glycoprotein (a 2-HSG) has been described (Pillai S., Zhou G. X., Arnaud P., Jiang H., Butler W. J. & Zhang H. (1996) Antibodies to endometrial transferrin and alpha 2-Heremans Schmidt (HS) glycoprotein in patients with endometriosis [published erratum appears in Am J Reprod Immunol 1997 March; 37(3):277]. Am J Reprod Immunol, 35, 483 and Mathur S. P., Holt V. L., Lee J. H., Jiang H. & Rust P. F. (1998) Levels of antibodies to transferrin and alpha 2-HS glycoprotein in women with and without endometriosis. Am J Reprod Immunol, 40, 69) and proposed as diagnostic markers.
Sialic acid-rich glycoproteins (sialoglycoproteins) bind selectin in humans and other organisms. Metastatic cancer cells often express a high density of sialic acid-rich glycoproteins. One example of the use of our methods and device may be in the diagnosis of melanoma. Melanoma is a malignant cancer of the skin. It is the eighth most common cancer in the United States and causes 1 to 2 percent of all cancer deaths. The diagnosis of melanoma is made on the basis of an excisional biopsy. However, not all excisional biopsies result in the diagnosis of melanoma. Also, recurrent nevus may be interpreted erroneously as a melanoma. HMB-45 is an antibody widely used in diagnostic pathology owing to its great specificity in identifying melanomas. Sialylation of antigen is crucial to HMB-45 binding, and suggests that the absence of staining in normal adult melanocytes, dermal nevi, and other melanocytic lesions may be a result of differential sialylation. Similarly, the differential detection of non-sialated and sialated proteins may aid in the rapid diagnosis of breast cancer, oral cancer, Alzheimer's disease and similar sialated/non-sialated protein or tau-protein diseases.
The present invention satisfies a need in the art by providing devices and methods for sensitive, specific and rapid detection of non-sialated protein in a patient sample using microfluidics technology.